Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method

Phosphatidic Acid and Lysophosphatidic Acid Induce Haptotactic Migration of Human Monocytes

The present study was aimed at defining the chemotactic activity of phosphatidic acid, which is rapidly produced by phagocytes in response to chemotactic agonists. Exogenously added phosphatidic acid induced human monocyte directional migration across polycarbonate filters with an efficacy (number of cell migrated) comparable to that of "classical" chemotactic factors. In lipid specificity studies, activity of phosphatidic acid decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain with the most active form being the natural occurring 18:0,20:4-phosphatidic acid. Lysophosphatidic acid was also active in inducing monocyte migration. No other phospholipid and lysophospholipid tested was effective in this response. Monocyte migration was regulated by a gradient of phosphatidic acid and lysophosphatidic acid bound to the polycarbonate filter, in the absence of detectable soluble chemoattractant. Migration was also observed if phospholipids were bound to fibronectin-coated polycarbonate filters. Thus, phosphatidic acid and lysophosphatidic acid, similarly to other physiological chemoattractants (e.g. C5a and interleukin-8), induce cell migration by an haptotactic mechanism. Phosphatidic acid caused a rapid increase of filamentous actin and, at higher concentrations, induced a rise of intracellular calcium concentration. Monocyte migration to phosphatidic acid and lysophosphatidic acid, but not to diacylglycerol, was inhibited in a concentration-dependent manner by Bordetella pertussis toxin, while cholera toxin was ineffective. In the chemotactic assay, phosphatidic acid and lysophosphatidic acid induced a complete homologous desensitization and only partially cross-desensitized one with each other, or with diacyl-glycerol and monocyte chemotactic protein-1. Suramine inhibited monocyte chemotaxis with a different efficiency phosphatidic acid > lysophosphatidic acid" diacyl-glycerol On the contrary, monocyte chemotactic protein-1-induced chemotaxis was not affected by the drug. Collectively, these data show that phosphatidic acid induces haptotactic migration of monocytes that is at least in part receptor-mediated. These results support a role for phosphatidic acid and lysophosphatidic acid in the regulation of leukocyte accumulation into tissues

Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study

In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

ABSTRACT Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates

Molecular cloning and functional characterization of brefeldin A-ADP-ribosylated substrate. A novel protein involved in the maintenance of the Golgi structure.

Brefeldin A (BFA) is a fungal metabolite that disassembles the Golgi apparatus into tubular networks and causes the dissociation of coatomer proteins from Golgi membranes. We have previously shown that an additional effect of BFA is to stimulate the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa (brefeldin A-ADP-riboslyated substrate (BARS)) and that this effect greatly facilitates the Golgi-disassembling activity of the toxin. In this study, BARS has been purified from rat brain cytosol and microsequenced, and the BARS cDNA has been cloned. BARS shares high homology with two known proteins, C-terminal-binding protein 1 (CtBP1) and CtBP2. It is therefore a third member of the CtBP family. The role of BARS in Golgi disassembly by BFA was verified in permeabilized cells. In the presence of dialyzed cytosol that had been previously depleted of BARS or treated with an anti-BARS antibody, BFA potently disassembled the Golgi. However, in cytosol complemented with purified BARS, or even in control cytosols containing physiological levels of BARS, the action of BFA on Golgi disassembly was strongly inhibited. These results suggest that BARS exerts a negative control on Golgi tubulation, with important consequences for the structure and function of the Golgi complex

the importance of an early alert from the microbiology laboratory and multidisciplinary collaboration during a suspected salmonellosis outbreak

Background and aims. Salmonellosis is one of the most common and widely distributed food-borne diseases. The increasing complexity and globalization of the food industry are causing an increase of some of these large-scale food-borne illnesses, thus there is a need for improvements in public health signal detection and communication streams between laboratories and regulatory agencies. The aim of this study is to show how the early reporting of salmonellosis cases directly from the Laboratory of Microbiology to the Local Health Service Infectious Diseases Office along with the prompt response of the ASL, and the rapid involvement of the Local Veterinary Prevention Department resulted in an improved individuation and investigation of a suspected food-borne outbreak with anomalous manifestation. Materials and methods. From August to November 2014 the early warning from the Laboratory of Microbiology regarding Salmonella spp. isolates with the identical serogroup and antibiotic resistance phenotype, allowed for prompt identification of a food-borne infection. Results and conclusions. The genotyping analysis suggested that over the period considered there was more than a single monophasic Salmonella typhimurium isolate: one responsible for the sporadic cases that occurred in September and October, and another in November

<i>Besnoitia besnoiti</i> among cattle in insular and northwestern Italy: endemic infection or isolated outbreaks?

Background: Bovine besnoitiosis, caused by the apicomplexan Besnoitia besnoiti, is a chronic and debilitating disease considered as emerging in Europe. In Spain, Portugal and France it is endemic and foci of infection were recorded in Germany, Switzerland, Hungary, Greece and Italy. In Italy, cases of bovine besnoitiosis were registered both in imported and autochthonous cattle, and mostly in central regions; high seroprevalence was also revealed by an epidemiological survey performed in the southern part of the country. Aiming to update information on the disease in northwestern and insular areas of Italy, where data on bovine besnoitiosis were missing, a serosurvey was designed for the present study. Methods: Three thousand one hundred and forty bovine blood samples from both dairy and beef farms (n = 126) were collected in northwestern regions (Lombardy, Piedmont and Liguria) and in the island of Sardinia. Samples were analyzed by a standardized in-house ELISA and those resulted positive were re-tested by Western Blot (WB) for confirmation. On results obtained by both ELISA and WB, apparent (AP) and true prevalence (TP) were calculated at individual and herd levels. Further, a panel of sera resulted positive to ELISA was analyzed by IFAT. Results: A total of 712 animals (AP = 22.7%; TP = 18.8%) and 109 farms (AP = 86.5%; TP = 88.2%) showed a positive reaction in ELISA. Only ten (AP = 0.3%; TP = 0%) specimens proceeding from five farms (AP = 3.9%; TP = 1.7%) from Lombardy were confirmed positive to the WB, corresponding to two Holstein Friesian cows and eight beef cattle. IFAT showed a low sensitivity (44.4%) scoring positive in only four samples out of 9 positive to WB. Conclusions: The survey demonstrated that bovine besnoitiosis cannot still be considered endemic in whole Italy. In fact, independent foci of infection were registered only in Lombardy region. Therefore, a sanitary strategy aimed to increase control measures and to organize monitoring plans, by adequate diagnostic tools is necessary to avoid overestimation of B. besnoiti in Italy.</br

Inventory of available data and data sources and proposal for data collection on vector‐borne zoonoses in animals

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